Protective roles of highly conserved motif 1 in tardigrade cytosolic-abundant heat soluble protein in extreme environments.

Tardigrades are remarkable microscopic animals that survive harsh conditions such as desiccation and extreme temperatures. Tardigrade-specific intrinsically disordered proteins (TDPs) play an essential role in the survival of tardigrades in extreme environments. Cytosolic-abundant heat soluble (CAHS) protein, a key TDP, is known to increase desiccation tolerance and to protect the activity of several enzymes under dehydrated conditions. However, the function and properties of each CAHS domain have not yet been elucidated in detail. Here, we aimed to elucidate the protective role of highly conserved motif 1 of CAHS in extreme environmental conditions. To examine CAHS domains, three protein constructs, CAHS Full (1-229), CAHS ∆Core (1-120_184-229), and CAHS Core (121-183), were engineered. The highly conserved CAHS motif 1 (124-142) in the CAHS Core formed an amphiphilic α helix, reducing the aggregate formation and protecting lactate dehydrogenase activity during dehydration-rehydration and freeze-thaw treatments, indicating that CAHS motif 1 in the CAHS Core was essential for maintaining protein solubility and stability. Aggregation assays and confocal microscopy revealed that the intrinsically disordered N- and C-terminal domains were more prone to aggregation under our experimental conditions. By explicating the functions of each domain in CAHS, our study proposes the possibility of using engineered proteins or peptides derived from CAHS as a potential candidate for biological applications in extreme environmental stress responses.

Immobilization protects enzymes from plasma-mediated inactivation.

Non-thermal plasmas are used in various applications to inactivate biological agents or biomolecules. A complex cocktail of reactive species, (vacuum) UV radiation and in some cases exposure to an electric field together cause the detrimental effects. In contrast to this disruptive property of technical plasmas, we have shown previously that it is possible to use non-thermal plasma-generated species such as H2O2 as cosubstrates in biocatalytic reactions. One of the main limitations in plasma-driven biocatalysis is the relatively short enzyme lifetime under plasma-operating conditions. This challenge could be overcome by immobilizing the enzymes on inert carrier materials. Here, we tested whether immobilization is suited to protect proteins from inactivation by plasma. To this end, using a dielectric barrier discharge device (PlasmaDerm), plasma stability was tested for five enzymes immobilized on ten different carrier materials. A comparative analysis of the treatment times needed to reduce enzyme activity of immobilized and free enzyme by 30% showed a maximum increase by a factor of 44. Covalent immobilization on a partly hydrophobic carrier surface proved most effective. We conclude from the study, that immobilization universally protects enzymes under plasma-operating conditions, paving the way for new emerging applications.

A simple method to purify intrinsically disordered proteins by adjusting trichloroacetic acid concentration.

Late embryogenic abundant proteins (LEA) are a group of proteins that accumulate during the desiccation phase of the seed and in response to water deficit in the plant. Most LEA proteins are highly hydrophilic and have physicochemical characteristics similar to those of intrinsically disordered proteins (IDPs). Although the function of LEA proteins is not fully understood, there is evidence indicating that these proteins have an important role in reducing the effects caused by water limitation. The analysis of the biochemical and physicochemical characteristics of LEA proteins is crucial to determine their function, for which it is necessary to obtain large amounts of pure protein. Within this current work, we have improved our previous TCA purification method used for basic recombinant LEA proteins to obtain acidic IDPs, the method reported here is fast and simple and is based on the enrichment of the protein of interest by boiling of the bacterial extract followed by a precipitation with different concentrations of TCA and salt. This protocol was applied to acidic and basic IDPs, represented by eight recombinant LEAs, resulting in milligram quantities of highly enriched proteins, which keep their in vitro functionality.

Synthetic Virus-like Particles for Glutathione Biosynthesis.

Protein-based nanocompartments found in nature have inspired the development of functional nanomaterials for a range of applications including delivery of catalytic activities with therapeutic effects. As glutathione (GSH) plays a vital role in metabolic adaptation and many diseases are associated with its deficiency, supplementation of GSH biosynthetic activity might be a potential therapeutic when delivered directly to the disease site. Here, we report the successful design and production of active nanoreactors capable of catalyzing the partial or complete pathway for GSH biosynthesis, which was realized by encapsulating essential enzymes of the pathway inside the virus-like particle (VLP) derived from the bacteriophage P22. These nanoreactors are the first examples of nanocages specifically designed for the biosynthesis of oligomeric biomolecules. A dense packing of enzymes is achieved within the cavities of the nanoreactors, which allows us to study enzyme behavior, in a crowded and confined environment, including enzymatic kinetics and protein stability. In addition, the biomedical utility of the nanoreactors in protection against oxidative stress was confirmed using an in vitro cell culture model. Given that P22 VLP capsid was suggested as a potential liver-tropic nanocarrier in vivo, it will be promising to test the efficacy of these GSH nanoreactors as a novel treatment for GSH-deficient hepatic diseases.

Oncolytic Nanoreactors Producing Hydrogen Peroxide for Oxidative Cancer Therapy.

In situ generation of anticancer agents at the place of the disease is a new paradigm for cancer therapy. The production of highly potent drugs by nanoreactors through a facile synthesis pathway is demanded. We report an oncolytic nanoreactor platform loaded with the enzyme glucose oxidase (GOX) to produce hydrogen peroxide. For the first time, we realized a core-shell structure with encapsulated GOX under mild synthetic conditions, which ensured high remaining activity of GOX inside of the nanoreactor. Moreover, the nanoreactor protected the loaded GOX from proteolysis and contributed to increased thermal stability of the enzyme. The nanoreactors were effectively taken up into different cancer cells, in which they produced hydrogen peroxide by consuming intracellular glucose and oxygen, thereby leading to effective death of the cancer cells. In summary, our robust nanoreactors are a promising platform for effective anticancer therapy and sustained enzyme utilization.

A sulfonated mesoporous silica nanoparticle for enzyme protection against denaturants and controlled release under reducing conditions.

Mesoporous silica nanoparticles (MSiNPs) are attractive enzyme hosts, but current MSiNPs are limited by leaching, poor enzyme stabilization/protection, and difficulty in controlling enzyme release. Sulfonated MSiNPs are promising alternatives, but are challenged by narrow channels, lack of control over enzyme adsorption to particle surfaces, and controlled release of enzyme. By introducing amines on particle surfaces and sulfonate groups into the channels via disulfide bonds, we developed a unique sulfonated MSiNP to selectively encapsulate enzymes to the channels with enhanced enzyme stabilization under denaturing conditions. Via pore-expansion, the channel diameter was increased which allows for encapsulating nm-sized enzymes. This new concept/strategy to immobilize and deliver enzymes or other biomacromolecules were demonstrated using two model enzymes. Furthermore, we combine site-directed spin labeling with Electron Paramagnetic Resonance to confirm the enhanced enzyme-host interaction and reveal the preferred enzyme orientation in the channels. Lastly, the presence of disulfides allows for enzyme release under reducing conditions, a great potential for cancer treatments. To the best of our knowledge, this is the first report of sulfonated MSiNPs that simultaneously offer enhanced stability against denaturants and controlled release of enzymes under reducing conditions, with enzyme orientation resolved in the channels.

Y2SK2 and SK3 type dehydrins from Agapanthus praecox can improve plant stress tolerance and act as multifunctional protectants.

Two dehydrins from Agapanthus praecox (ApY2SK2 and ApSK3) show important protective effects under complex stresses. Both ApY2SK2 and ApSK3 contain one intron and consist of a full-length cDNA of 981 bp and 1057 bp encoding 186 and 215 amino acids, respectively. ApY2SK2 and ApSK3 transgenic Arabidopsis thaliana show reduced plasma membrane damage and ROS levels and higher antioxidant activity and photosynthesis capability under salt, osmotic, cold and drought stresses compared with the wild-type. ApY2SK2 and ApSK3 are mainly located in the cytoplasm and cell membrane, and ApY2SK2 can even localize in the nucleus. In vitro tests indicate that ApY2SK2 and ApSK3 can effectively protect enzyme activity during the freeze-thaw process, and ApY2SK2 also exhibits this function during desiccation treatment. Furthermore, ApY2SK2 and ApSK3 can significantly inhibit hydroxyl radical generation. These two dehydrins can bind metal ions with a binding affinity of Co2+> Ni2+> Cu2+> Fe3+; the binding affinity of ApSK3 is higher than that of ApY2SK2. Thus, ApY2SK2 has a better protective effect on enzyme activity, and ApSK3 has stronger metal ion binding function and effect on ROS metabolism. Moreover, plant cryopreservation evaluation tests indicate that ApY2SK2 and ApSK3 transformation can enhance the seedling survival ratio from 23% to 47% and 55%, respectively; the addition of recombinant ApY2SK2 and ApSK3 to plant vitrification solution may increase the survival ratio of wild-type A. thaliana seedlings from 24% to 50% and 46%, respectively. These findings suggest that ApY2SK2 and ApSK3 can effectively improve cell stress tolerance and have great potential for in vivo or in vitro applications.

Protecting activity of desiccated enzymes.

Protein-based biological drugs and many industrial enzymes are unstable, making them prohibitively expensive. Some can be stabilized by formulation with excipients, but most still require low temperature storage. In search of new, more robust excipients, we turned to the tardigrade, a microscopic animal that synthesizes cytosolic abundant heat soluble (CAHS) proteins to protect its cellular components during desiccation. We find that CAHS proteins protect the test enzymes lactate dehydrogenase and lipoprotein lipase against desiccation-, freezing-, and lyophilization-induced deactivation. Our data also show that a variety of globular and disordered protein controls, with no known link to desiccation tolerance, protect our test enzymes. Protection of lactate dehydrogenase correlates, albeit imperfectly, with the charge density of the protein additive, suggesting an approach to tune protection by modifying charge. Our results support the potential use of CAHS proteins as stabilizing excipients in formulations and suggest that other proteins may have similar potential.

Alternative Enzyme Protection Assay To Overcome the Drawbacks of the Gentamicin Protection Assay for Measuring Entry and Intracellular Survival of Staphylococci.

Precise enumeration of living intracellular bacteria is the key step to estimate the invasion potential of pathogens and host immune responses to understand the mechanism and kinetics of bacterial pathogenesis. Therefore, quantitative assessment of host-pathogen interactions is essential for development of novel antibacterial therapeutics for infectious disease. The gentamicin protection assay (GPA) is the most widely used method for these estimations by counting the CFU of intracellular living pathogens. Here, we assess the longstanding drawbacks of the GPA by employing an antistaphylococcal endopeptidase as a bactericidal agent to kill extracellular Staphylococcus aureus We found that the difference between the two methods for the recovery of intracellular CFU of S. aureus was about 5 times. We prove that the accurate number of intracellular CFU could not be precisely determined by the GPA due to the internalization of gentamicin into host cells during extracellular bacterial killing. We further demonstrate that lysostaphin-mediated extracellular bacterial clearance has advantages for measuring the kinetics of bacterial internalization on a minute time scale due to the fast and tunable activity and the inability of protein to permeate the host cell membrane. From these results, we propose that accurate quantification of intracellular bacteria and measurement of internalization kinetics can be achieved by employing enzyme-mediated killing of extracellular bacteria (enzyme protection assay [EPA]) rather than the host-permeative drug gentamicin, which is known to alter host physiology.

Protecting bactofencin A to enable its antimicrobial activity using mesoporous matrices.

There is huge global concern surrounding the emergence of antimicrobial resistant bacteria and this is resulting in an inability to treat infectious diseases. This is due to a lack of new antimicrobials coming to the market and irresponsible use of traditional antibiotics. Bactofencin A, a novel antimicrobial peptide which shows potential as an antibiotic, is susceptible to enzyme degradation. To improve its solution stability and inherent activity, bactofencin A was loaded onto a traditional silica mesoporous matrix, SBA-15, and a periodic mesoporous organosilane, MSE. The loading of bactofencin A was considerably higher onto SBA-15 than MSE due to the hydrophilic nature of SBA-15. While there was no detectable peptide released from SBA-15 into phosphate buffered saline and only 20% of the peptide loaded onto MSE was released, the loaded matrices showed enhanced activity compared to the free peptide during in vitro antimicrobial assays. In addition, the mesoporous matrices were found to protect bactofencin A against enzymatic degradation where results showed that the SBA-15 and MSE with loaded bactofencin A exposed to trypsin inhibited the growth of S. aureus while a large decrease in activity was observed for free bactofencin upon exposure to trypsin. Thus, the activity and stability of bactofencin A can be enhanced using mesoporous matrices and these matrices may enable its potential development as a novel antibiotic. This work also shows that in silico studies looking at surface functional group and size complementarity between the peptide and the protective matrix could enable the systemic selection of a mesoporous matrix for individual bacteriocins with potential antimicrobial therapeutic properties.

Human MMAA induces the release of inactive cofactor and restores methylmalonyl-CoA mutase activity through their complex formation.

Human mitochondrial methylmalonyl-CoA mutase (hMCM) is an isomerase that converts methylmalonyl-CoA to succinyl-CoA, a crucial step for the incorporation of some compounds derived from the diet into the central metabolism. hMCM employs highly reactive radicals from its cofactor (adenosylcobalamin, AdoCbl) to perform its reaction. Our previous work demonstrated that hMCM loses activity during catalysis and that the interaction with human MMAA (hMMAA), a GTPase protein, avoided this loss or restored hMCM activity. Even so, the mechanism by which hMMAA exerted these chaperone functions has not been described. In this work report that the formation and accumulation of OH2Cbl, the oxidized form of the AdoCbl cofactor formed during catalysis, is the cause of hMCM inactivation. Additionally, we demonstrate that the complex formation of hMCM/hMMAA decreases the rate of oxidized cofactor formation, protecting the hMCM enzyme. Moreover, an inactive model of hMCM was used to demonstrate that hMMAA is able to remove the damaged cofactor through GTP hydrolysis. Additionally, a modification in the kinetic parameters of hMCM in presence of hMMAA was observed, and for the first time, the in vivo localization of hMMAA and its colocalization with hMCM in human fibroblasts mitochondria were demonstrated.